Mass spectrometry has been used to determine the extent of modification and the specific sites of modification on biomolecules. MS-based approaches have many advantages, including generally rapid analyses without radiolabeling. The MS analysis of protein-DNA crosslinks has been investigated using mass spectrometry. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and LC in combination with electrospray mass spectrometry. Additionally, products following crosslinking have been purified by SDS-PAGE and investigated by mass spectrometry. With the addition of a cryo-electron microscopy (cryo-EM) facility, many PIs at NIEHS will be incorporating this technique into their studies. Publications show that this technique is often paired with crosslinking followed by mass spectrometric analyses. We are currently analyzing the use of the crosslinker BS3 with the proteins Grc3 and Las1 by mass spectrometry in collaboration with the Stanley laboratory to try to determine which residues from Grc3 and Las1 interact. Other work within the MSRSG in the characterization of biomolecules: Characterization of dust and proteins from allergens in collaboration - Don Cook, Geoffrey Mueller, and Robert London Characterization of protein:protein cross-links in DNA repair enzymes and characterization of sites of ubiquitination and sumoylation in DNA repair enzymes Scott Williams Characterization of phosphorylation sites on the serine/threonine kinase VRK1 Masahiko Negishi Recently published work in collaboration with Drs. Kunkel and Pedersen that falls under this heading focuses on the characterization of adenylation on DNA Ligase IV. DNA ligase IV (LigIV) performs the final DNA nick-sealing step of classical nonhomologous end-joining, which is critical for immunoglobulin gene maturation and efficient repair of genotoxic DNA double-strand breaks. Hypomorphic LigIV mutations cause extreme radiation sensitivity and immunodeficiency in humans. To better understand the unique features of LigIV function, here we report the crystal structure of the catalytic core of human LigIV in complex with a nicked nucleic acid substrate in two distinct statesan open lysyl-AMP intermediate, and a closed DNAadenylate form. Results from structural and mutagenesis experiments unveil a dynamic LigIV DNA encirclement mechanism characterized by extensive interdomain interactions and active site phosphoanhydride coordination, all of which are required for efficient DNA nick sealing. These studies provide a scaffold for defining impacts of LigIV catalytic core mutations and deficiencies in human LIG4 syndrome. The specific role of the MSRSG was to identify adenylation on LigIV. Other recently published work in collaboration with the Stanley laboratory focuses on the characterization of the role of the Las1/Grc3 complex. Las1 is a recently discovered endoribonuclease that collaborates with Grc3-Rat1-Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here, we report that the essential Las1 endoribonuclease requires its binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results establish that Grc3 drives Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively toward single-stranded RNA. Together, Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 cross talk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a unique example of a protein-guided programmable endoribonuclease. The specific role of the MSRSG was to characterize the enzymatic cleavage site on the RNA.
| Kaminski, Andrea M; Tumbale, Percy P; Schellenberg, Matthew J et al. (2018) Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis. Nat Commun 9:2642 |
| Pillon, Monica C; Sobhany, Mack; Borgnia, Mario J et al. (2017) Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage. Proc Natl Acad Sci U S A 114:E5530-E5538 |
